VOL. 11, NO. 4, FEBRUARY 2016
ISSN 1819-6608
ARPN Journal of Engineering and Applied Sciences
©2006-2016 Asian Research Publishing Network (ARPN). All rights reserved.
www.arpnjournals.com
THE EFFECT OF NITROGEN STRESS IN MEDIUMFOR INCREASING
CARBOHYDRATE AS A BIOETHANOL SOURCE AND CAROTENOID
AS AN ANTIOXIDANT FROM CHLORELLA ZOFINGIENSIS CULTURE
Eko Agus Suyono1,2, Umi Muavatun1,, Faridatul Husna1, Husnul Khotimah1, Ika Pratiwi1, Rahmah Husna1, Fitri
Cahyani1, Yuni Purwanti1 and Thoriq Teja Samudra1
1
Faculty of Biology, Gadjah MadaUniversity Jl. Teknika Selatan, Sekip Utara, Yogyakarta, Indonesia
Centre for Energy Studies, GadjahMada University Sekip K1A, Kampus UGM, Yogyakarta, Indonesia
E-Mail: [email protected]
2
ABSTRACT
Chlorella zofingiensis is a prospective microalgae because it is mainly ascarotenoid producer, such as astaxanthin.
However, its carbohydrates could be also as promising source ofbioethanol. Furthermore, Nitrogen stress treatment is
reported used for increasing both carbohydrate and carotenoid of some microalgae. Therefore, this study aimed to increase
carbohydrate and carotenoid of microalgae C. zofingiensis by using low and high nitrogen excessin the growth medium.
The mediums were consisted of local compound fertilizer (farmpion), urea and ZA with a ratio of 0.25:0.5:1(low nitrogen
excess medium) and 0.5: 1: 2(high nitrogen excess medium). Its cells density, carotenoid, and carbohydrate were measured
every day for 7 days. The cell density was calculated using haemocytometer under light microscope. The carotenoid was
measured using spectrophotometer with absorbance at a wavelength of 470, 645 and 662 nm. The carbohydrate was
measured using sulfuric acid method. The results showed that the nitrogen stress treatment was able to increase
carbohydrates and carotenoids approximately twice in C. zofingiensis as culture as source of bioethanol and antioxidant.
Keywords: chlorella zofingiensi, nitrogen, carotenoid, carbohydrate.
INTRODUCTION
Chlorella zofingiens is freshwater green algal that
classified into class and family Chlorophyceae and ordo
Chlorococcales [1] and indicates having high carbohydrate
content as bioethanol source [2]. That product has been
proposed as good alternative to non-renewable fossil fuels
[3]. Microalgal is as a potential new generation of
feedstock for biofuel production because of its high
growthh rates and high photosynthetic efficiencies when
grown on specific environments [4].
When the microalgae is cultivated and grown in
extreme environmental conditions and under stress
circumstances, it will synthesize various secondary
metabolites to increase the possibility for surviving under
those stress conditions [5, 6]. One of the secondary
metabolites produced by the microalgae is carotenoid
asantioxidant property [7]. Microalgae C. zofingiensis is
reportedable accumulate the carotenod, especially
astaxhanthin as a king of antioxidant [8], so that that the
microalgae is potentially as astaxanthin producer [9].
Aprospective method for enhancing its valuable
contents is by modifying Nitrogen concentration in the
growth medium. Nitrogen is an essential nutrient for
increasing C. Zofingiens is carbohydrate and carotenoid
[10]. Nitrogen is used for producing chlorophyll which is
an important compound to accelerate the photosynthesis
rate.
Therefore, the aim of this research is to increase
carbohydrate and carotenoid of microalgae C. zofingiensis
by nitrogen excess treatments in the medium. The research
will be an alternative method for producing bioethanol
from high carbohydrate microalgae as a future renewable
energy as well as producing carotenoid as a valuable
antioxidant.
MATERIALS AND METHODS
Materials
This research used medium agricultural
fertilizers: urea: ZA with a ratio of 0.5: 1: 2 (high excess
nitrogen medium) and 0.25: 0, 5: 1(low excessnitrogen
medium).
Method
The study was conducted in Wukir Sari,
Cangkringan, Pakem, Sleman, Daerah Istimewa
Yogyakarta (DIY). C. zofingiensis was cultured on a mass
scale in the 3600 litre pool. Environmental parameters
measured were temperature, pH, and density. The
mediums were local agricultural fertilizer (farmpion), urea
and ZA with ratio of 0.25: 0, 5: 1 (low excess nitrogen
medium) and 0, 5: 1: 2 (high excess nitrogen medium). As
a control, C. zofingiensis was cultivated in medium with
local agricultural fertilizer (farmpion) without the addition
of urea and ZA. Nitrogen content in fertilizers and ZA
21%, while the nitrogen contentinUrea46%. Samples were
taken every day for 7 days. The parameters measured were
cells density, carotenoid, and charbohydrate.
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VOL. 11, NO. 4, FEBRUARY 2016
ISSN 1819-6608
ARPN Journal of Engineering and Applied Sciences
©2006-2016 Asian Research Publishing Network (ARPN). All rights reserved.
www.arpnjournals.com
The Calculation of the Cells Density
Cell counting using Haemacytometer, samples
were taken 800μL inserted into the tube 2mL was added
200μL 70% alcohol, and wait about 20 minutes before
counted under a microscope. Calculations done by
counting cells with two fields of view on Haemacytometer
then calculated using the following formula:
ellsdensity
numberofcellscounted
cell
=
xdilutionfactorx 5 x
5
mL
Calculation of Carotenoids
Samples were taken 10 mL inserted into the tube,
then centrifuge at a speed of 3300 rpm for 15 minutes. The
supernatant was discharged; andthe sample was added by
2 mL of acetone, then centrifuged again with a rate of1800
rpm for 10 minutes. The sample was transferred into a
glass cuvette spectrophotometer and calculated by using
absorbance at a wavelength of 470, 645 and 662 nm. The
carotenoids could be calculated by using the following
equation [11].
��� �
�
�
�
=
�
−�
���
�
�
�
�
�
Calculation of Carbohydrates
Standard curve made used standard glucose, with
concentration of 0,025 g/l, 0,05 g/l, 0,1 g/l, 0.25 g/l and
0.5 g/l was observed by using a spectrophotometer with a
wavelength 0f 492 nm. The samples were taken and added
30 mL phenol and 150 mL sulfuric acid, then let stand for
30 minutes and the absorbance was measured using a
spectrophotometer with a wavelength of 490 nm.
RESULTS AND DISCUSSIONS
As a photosynthetic organism, C. zofingiensis has
pigments used for harvesting light energy. Chlorophyll
was the main pigment of photosynthesis. Chlorophyll was
composed of tetraphyrrole ring containing atoms of
magnesium, nitrogen and long chain terpenoids [12]. So
that the provision of nitrogen could increase the formation
of chlorophyll. Increased formation of chlorophyll could
increase the rate of photosynthesis and the more energy
was produced. Nitrogen was an important element in the
growth of microalgae. Nitrogen was an important nutrient
after the formation of carbon in biomass [13]. Nitrogen
was generally available in the form of nitrate (NO3-) and
ammonia (NH4+). Nitrogen sources in this experiment
were urea and ZA. Urea was a nitrogen source that could
provide CO2 for photosynthesis [14]. A high nitrogen
content that was 2-3 times higher than normal
concentration in the medium increased the synthesis of
chlorophyll pigments in cells [15].
When the chlorophyll content increased, the rate
of photosynthesis would also increase. If the process of
photosynthesis increased, the carbohydrates produced
from photosynthesis process would also increase.
Carbohydrates formed through the photosynthesis process,
most carbohydrates were used as energy through
respiration reaction [16]. While others were stored in
carbohydrate glandular or in the form of cellulose of cell
walls, to result in increased cell biomass [17].
Carbohydrates were used for energy sources. Energy
produced through photosynthesis was used to multiply the
number of cells and stored in the form of carbohydrates,
especially starch and cellulose [13].
In this study, increasing of carbohydrate
production in photosynthesis was as source of energy. The
carbohydrate could be used for bioethanol production to
conduct an alternative energy. Microalgae had the ability
to alter their biomass composition under stress conditions
to accumulate more carbohydrates. The carbohydrates
were broken into their sugars. These sugars could be
fermented to produce bioethanol [2].
As could be seen in Figure-1, the carbohydrate
per cells in both low and high excess nitrogen mediums
tend to increase. In all treatments, the carbohydrate per
cell decreased significantly at the day 5 as the number of
cells was at the pick. However, the trend increased
dramatically at the day 6 to 7. It was predicted that the cell
growth slowly, but the amount of carbohydrate still
increased at those days.This indicated that the nitrogen
concentration affected on the formation of carbohydrates
indirectly. The nitrogen as an element that forming the
light-harvesting chlorophyll accelerated photosynthesis
products accumulated in the formation of carbon biomass
[13].
Figure-1. (a) Carbohydrate total in C. zofingiensis (b)
Carbohydrate per cell content in C. zofingiensis
Control
Low nitrogen excess
High nitrogen excess.
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VOL. 11, NO. 4, FEBRUARY 2016
ISSN 1819-6608
ARPN Journal of Engineering and Applied Sciences
©2006-2016 Asian Research Publishing Network (ARPN). All rights reserved.
www.arpnjournals.com
Figure-2. (a) Total carotenoid in C. zofingiensis (b) Carotenoid per cell content in C. Zofingiensis
Control
Low nitrogen excess
High nitrogen excess.
Some microalgal species cultured under stress
conditions could accumulate specific secondary
metabolites, such as pigments, vitamins or lipids [18]. As
shown in Figure-2, the carotenoid per cell tend to increase,
but the ratio of carbohydrates to carotenoids tend to
decline (Figure-3) in both low and high nitrogen excess
mediums. It was assumed that both treatments tend to
form more carotenoids than carbohydrate. Carotenoids
were pigments that were involved in the light harvesting
reaction and protection of algal cellorganelle against single
Synthesis of astaxanthin required nitrogen, and
most likely reflected the need for continuous synthesis of
protein in order to support the massive accumulation of
pigments [28]. Nitrogen was essential element for de novo
synthesis of enzymes responsible for carotenoid formation
[27]. The nitrogen stress could also increase the
carbohydrate content in the cells. The carbohydrate could
be broken to be sugars and fermented to produce
bioethanol [2] and the carotenoid could be used as
antioxidant. It could be concluded that both low and high
nitrogen increased carbohydrate and carotenoid content in
C. Zofingiensis.
REFERENCES
[1] Tripathi, U., Sarada, R., Rao, S. and Ravishankar, G.
1999.
Production
of
astaxan
thin
in
Haematococcuspluvialis cultured in various media.
Bioresour. Technol. 68: 197-199.
Figure-3. Carbohydrate per carotenoidtotal in C.
zofingiensis
Control
Low nitrogen excess
High nitrogen excess.
Oxygen damage [19]. Carotenoids in green algal were
produced in two different pathways, i.e. acetic-mevalonate
pathway and phosphegly ceraldehyde-pyruvate pathway
[20, 21]. More carotenoids produced due to environmental
stress or adverse culture conditions such nutrient excess
and starvation [22]. The nitrogen stress stimulated the
formation of carotenoids, especially astaxan thin [23].One
of
the
studies
that
have
been
done
on
Haematococcuspluvial is also showed that heantioxidant
pigment astaxan thin produced in a variety of stress
conditions such as high light, salinity, nutrient stress, the
concentration of carbon or high nitrogen [24, 25, 26]. It
could be clearly stated that nitrogen was an important
nutrient for biomass production [13] and carotenoid
production [27].
[2] Campo J, García-González M, Guerrero M.2007.
Outdoor cultivation of microalgae for carotenoid
production: current state and perspectives. Appl
Microbiol. Biotechnol. 74: 1163–74.
[3] Ladygin. V. G. 2000. Biosynthesis of carotenoids in
the chloroplasts of algae and higher plants. Russ. J.
Plant Physiol.47, 904-923.
[4] Sheehan, J., Dunahay, T., Benemann, J. and
Roessler,P. 1998. Look Backatthe U.S. Department of
Energy’s Aquatic Species Program: Biodiesel from
Algae. Golden: National Renewable Energy
Laboratory.
[5] Klein-Marcuschamer D., Chisti Y., Benemann J. R,
Lewis D. A. 2013. Matter of detail: assessing the true
potential of microalgal biofuels. Biotechnol .Bioeng.
110:2317-22.
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VOL. 11, NO. 4, FEBRUARY 2016
ISSN 1819-6608
ARPN Journal of Engineering and Applied Sciences
©2006-2016 Asian Research Publishing Network (ARPN). All rights reserved.
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[6] Baushiba. S. and Vonshak. A. 1991. Astaxanthin
accumulation in the green alga Haematococcis
pluvialis. Plant cell physiol. 32: 1077-1082.
[18] Pickett-Heaps. J. D. 1975. Green algae: structure,
reproduction and evolution in selected genera. Sinauer
Associates, pp.72.
[7] Del Campo JA, Rodríguez H, Moreno J, Vargas MÁ,
Rivas J, Guerrero MG.2004. Accumulation of
astaxanthin and lutein in Chlorella zofingiensis
(Chlorophyta). Appl. Microbiol. Biotechnol. 64:84854.
[19] Weil. C.M. 2000. Exploring Hopein Patient with End
Stage Renal Diseaseon Chronic Hemodialysis. ANNA
Journal. 27: 219-223.
[8] Carmichael W.W.1992 Cyanobacteria secondary
metabolites
the
cyanotoxins.
J.
Appl.
Microbiol72:445–59.
[9] Berman. T. and Chava. S. 1999. Algal growth on
organic compounds as nitrogen source. Journal of.
Plankton. Res. 21 (8): 1423-37.
[10] Skjånes K, Rebours C, Lindblad P.2012. Potential for
green
microalgae
to
produce
hydrogen,
pharmaceuticals and other high value products in a
combined process. Crit. Rev. Biotechnol. 1-44.
[11] Guedes. A.C. and Malcata. F.X. 2012. Nutritional
Value and Uses of Microalgae in Aquaculture. J. Aq.
Culture. 4: 61-78
[12] Taiz. L.and Zeiger. E. 2002. Plant Physiology. 3rd
Edition. Sunderland: Sinauer Associates. pp. 116-119.
[13] Marchetti, J., G. Bougaran. T. Jauffrais. S. Lefebvre.
C. Rouxel. B. Saint-Jean. E. Lukomska. R. Robert.and
J.P. Cadoret. 2012. Effects of blue light on [14] Pisal,
D. S. And Lele, S. S. 2005.Carotenoidproduction
from microalga Dunaliellasalina.Indian J. Biotech.
4:476-483.
[14] Corsini. N. and Karydis. M. 1990. An algal medium
best and fertilizers and its evaluation in mariculture.
Journal of Appl. Phycol. 2:333-339.
[15] Sarada, R., Tripathi, U., and Ravishankar, G. 2002.
Influence of stress on astaxan thin production in
Haematococcuspluvial is grown under different
culture conditions. J Process Biochem. 37: 623-627.
[16] Cunningham.F. X. 2002. Regulation of carotenoid
synthesis and accumulation in plants.Pure. Appl.
Chem.74: 1409-1417.
[17] Dere, S. Tohit, G. and Ridvan. S. 1998.
Spectrophotometric Determination of Chlorophyll-A,
B, and Total Carotenoid Contens of Some Algae
Species Using Different Solvent. J. Bot. 22: 13-17.
[20] Richmond. A. 2004. Handbook of microalgal culture:
biotechnology and applied phycology. Blackwell
science Ltd. UK. pp. 3; 105. the biochemical
composition and photosynthetic activity of
Isochrysissp. (T-iso). J. Appl. Phycol. 6: 756-765.
[21] Becker. E.W. 1994. Microalgae: biotechnology and
microbiology.
Cambridge
studies
in
biotechnology.Cambridge
university
press.
Cambridge. p. 104.
[22] Johnson. E. A. and Schroeder. W. A. 1996. Microbial
carotenoids. Advance. Biochem. 53:119-178.
[23] Kakizono. T. Kobayashi. M. and Nagai. S. 1992.
Effect of carbon/nitrogen ratio on encystment
accompanied with astaxan thin formation in a green
alga, Haematococcus pluvialis. Journal of Ferm and
Bioeng. 74: 403-405.
[24] Ho.S.H. Huang. S.W. Chen. C. Y.Hasunuma. T.,
Kondo. A. Chang. J. S. 2013. Bioethanol production
using carbohydrate-rich microalgae biomass as
feedstock. Biores. Tech., 135: 191-8.
[25] Suyono, E.A., Aminin, Pradani, L., Mu’avatuna, U.,
Habiba, R, N., Ramdaniya, Rohma, E.F., 2015.
Combination of blue, red, white, and ultraviolet lights
for increasing carotenoids and biomass of Microalga
Haematococcuspluvialis. Procedia Environ Sci. 28:
399-405.
[26] El-Sayed. A. B. 2010. Carotenoid accumulation in the
green alga Scenedesmus sp. incubated with industrial
citrate waste and differentinduction stresses. Nat and
Sci. 8 (10): 37.
[27] Skjanes, K., Rebours, C. and Lindblad, P. 2013.
Potential for green microalgae to produce hydrogen,
pharmaceuticals and other high value products in a
combined process. Crit. Rev. Biotechnol. 33: 172215.
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